Successful transport across continents of GMP-manufactured and cryopreserved culture-expanded human fetal liver-derived mesenchymal stem cells for use in a clinical trial.

Introduction: Cell therapy has been increasingly considered to treat diseases, but it has been proven difficult to manufacture the same product at multiple manufacturing sites. Thus, for a wider implementation an alternative is to have one manufacturing site with a wide distribution to clinical sites. To ensure administration of a good quality cell therapy product with maintained functional characteristics, several obstacles must be overcome, which includes for example transfer of knowledge, protocols and procedures, site assessment, transportation and preparation of the product. Methods: As the preparatory work for a clinical trial in India using fetal mesenchymal stem cells (fMSCs) developed and manufactured in Sweden, we performed a site assessment of the receiving clinical site, transferred methods, developed procedures and provided training of operators for handling of the cell therapy product. We further developed a Pharmacy Manual to cover the management of the product, from ordering it from the manufacturer, through transport, reconstitution, testing and administration at the clinical site. Lastly, the effect of long-distance transport on survival and function of, as well as the correct handling of the cell therapy product, was evaluated according to the pre-determined and approved Product Specification. Results: Four batches of cryopreserved human fetal liver-derived fMSCs manufactured according to Good Manufacturing Practice and tested according to predetermined release criteria in Sweden, were certified and transported in a dry shipper at -150 °C to the clinical site in India. The transport was temperature monitored and took three-seven days to complete. The thawed and reconstituted cells showed more than 80% viability up to 3 h post-thawing, the cell recovery was more than 94%, the cells displayed the same surface protein expression pattern, differentiated into bone, had stable chromosomes and were sterile, which conformed with the data from the manufacturing site in Sweden. Conclusions: Our study shows the feasibility of transferring necessary knowledge and technology to be able to carry out a clinical trial with a cell therapy product in distant country. It also shows that it is possible to transport a cryopreserved cell therapy product over long distances and borders with retained quality. This extends the use of cryopreserved cell therapy products in the future.

An exploratory open-label multicentre phase I/II trial evaluating the safety and efficacy of postnatal or prenatal and postnatal administration of allogeneic expanded fetal mesenchymal stem cells for the treatment of severe osteogenesis imperfecta in infants and fetuses: the BOOSTB4 trial protocol.

INTRODUCTION: Severe osteogenesis imperfecta (OI) is a debilitating disease with no cure or sufficiently effective treatment. Mesenchymal stem cells (MSCs) have good safety profile, show promising effects and can form bone. The Boost Brittle Bones Before Birth (BOOSTB4) trial evaluates administration of allogeneic expanded human first trimester fetal liver MSCs (BOOST cells) for OI type 3 or severe type 4. METHODS AND ANALYSIS: BOOSTB4 is an exploratory, open-label, multiple dose, phase I/II clinical trial evaluating safety and efficacy of postnatal (n=15) or prenatal and postnatal (n=3, originally n=15) administration of BOOST cells for the treatment of severe OI compared with a combination of historical (1-5/subject) and untreated prospective controls (≤30). Infants<18 months of age (originally<12 months) and singleton pregnant women whose fetus has severe OI with confirmed glycine substitution in COL1A1 or COL1A2 can be included in the trial.Each subject receives four intravenous doses of 3×106/kg BOOST cells at 4 month intervals, with 48 (doses 1-2) or 24 (doses 3-4) hours in-patient follow-up, primary follow-up at 6 and 12 months after the last dose and long-term follow-up yearly until 10 years after the first dose. Prenatal subjects receive the first dose via ultrasound-guided injection into the umbilical vein within the fetal liver (16+0 to 35+6 weeks), and three doses postnatally.The primary outcome measures are safety and tolerability of repeated BOOST cell administration. The secondary outcome measures are number of fractures from baseline to primary and long-term follow-up, growth, change in bone mineral density, clinical OI status and biochemical bone turnover. ETHICS AND DISSEMINATION: The trial is approved by Competent Authorities in Sweden, the UK and the Netherlands (postnatal only). Results from the trial will be disseminated via CTIS, ClinicalTrials.gov and in scientific open-access scientific journals. TRIAL REGISTRATION NUMBERS: EudraCT 2015-003699-60, EUCT: 2023-504593-38-00, NCT03706482.

Transcriptomic and proteomic profiles of fetal versus adult mesenchymal stromal cells and mesenchymal stromal cell-derived extracellular vesicles.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) can regenerate tissues through engraftment and differentiation but also via paracrine signalling via extracellular vesicles (EVs). Fetal-derived MSCs (fMSCs) have been shown, both in vitro and in animal studies, to be more efficient than adult MSC (aMSCs) in generating bone and muscle but the underlying reason for this difference has not yet been clearly elucidated. In this study, we aimed to systematically investigate the differences between fetal and adult MSCs and MSC-derived EVs at the phenotypic, RNA, and protein levels. METHODS: We carried out a detailed and comparative characterization of culture-expanded fetal liver derived MSCs (fMSCs) and adult bone marrow derived MSCs (aMSCs) phenotypically, and the MSCs and MSC-derived EVs were analysed using transcriptomics and proteomics approaches with RNA Sequencing and Mass Spectrometry. RESULTS: Fetal MSCs were smaller, exhibited increased proliferation and colony-forming capacity, delayed onset of senescence, and demonstrated superior osteoblast differentiation capability compared to their adult counterparts. Gene Ontology analysis revealed that fMSCs displayed upregulated gene sets such as "Positive regulation of stem cell populations", "Maintenance of stemness" and "Muscle cell development/contraction/Myogenesis" in comparison to aMSCs. Conversely, aMSCs displayed upregulated gene sets such as "Complement cascade", "Adipogenesis", "Extracellular matrix glycoproteins" and "Cellular metabolism", and on the protein level, "Epithelial cell differentiation" pathways. Signalling entropy analysis suggested that fMSCs exhibit higher signalling promiscuity and hence, higher potency than aMSCs. Gene ontology comparisons revealed that fetal MSC-derived EVs (fEVs) were enriched for "Collagen fibril organization", "Protein folding", and "Response to transforming growth factor beta" compared to adult MSC-derived EVs (aEVs), whereas no significant difference in protein expression in aEVs compared to fEVs could be detected. CONCLUSIONS: This study provides detailed and systematic insight into the differences between fMSCs and aMSCs, and MSC-derived EVs. The key finding across phenotypic, transcriptomic and proteomic levels is that fMSCs exhibit higher potency than aMSCs, meaning they are in a more undifferentiated state. Additionally, fMSCs and fMSC-derived EVs may possess greater bone forming capacity compared to aMSCs. Therefore, using fMSCs may lead to better treatment efficacy, especially in musculoskeletal diseases.

Maternal and fetal safety outcomes after in utero stem cell injection: A systematic review.

OBJECTIVE: To investigate the maternal and fetal safety of In utero stem cell transplantation (IUSCT). METHODS: Medline®, Embase and Cochrane library (1967-2023) search for publications reporting IUSCT in humans. Two reviewers independently screened abstracts and full-text papers. RESULTS: Sixty six transplantation procedures in 52 fetuses were performed for haemoglobinopathies (n = 14), red cell/bleeding disorders (n = 4), immunodeficiencies (n = 15), storage disorders (n = 7), osteogenesis imperfecta (n = 2) and healthy fetuses (n = 10). The average gestational age was 18.9 weeks; of procedures reporting the injection route, cells were delivered by intraperitoneal (n = 37), intravenous (n = 19), or intracardiac (n = 4) injection or a combination (n = 3); most fetuses received one injection (n = 41). Haematopoietic (n = 40) or mesenchymal (n = 12) stem cells were delivered. The cell dose was inconsistently reported (range 1.8-3.3 × 109 cells total (n = 27); 2.7-5.0 × 109 /kg estimated fetal weight (n = 17)). The acute fetal procedural complication rate was 4.5% (3/66); the acute fetal mortality rate was 3.0% (2/66). Neonatal survival was 69.2% (36/52). Immediate maternal and pregnancy outcomes were reported in only 30.8% (16/52) and 44.2% (23/52) of cases respectively. Four fetal/pregnancy outcomes would also classify as ≥ Grade 2 maternal adverse events. CONCLUSIONS: Short-, medium-, and long-term maternal and fetal adverse events should be reported in all IUSCT studies.

Regenerative medicine: prenatal approaches.

This two-paper Series focuses on recent advances and applications of regenerative medicine that could benefit paediatric patients. Innovations in genomic, stem-cell, and tissue-based technologies have created progress in disease modelling and new therapies for congenital and incurable paediatric diseases. Prenatal approaches present unique opportunities associated with substantial biotechnical, medical, and ethical obstacles. Maternal plasma fetal DNA analysis is increasingly adopted as a noninvasive prenatal screening or diagnostic test for chromosomal and monogenic disorders. The molecular basis for cell-free DNA detection stimulated the development of circulating tumour DNA testing for adult cancers. In-utero stem-cell, gene, gene-modified cell (and to a lesser extent, tissue-based) therapies have shown early clinical promise in a wide range of paediatric disorders. Fetal cells for postnatal treatment and artificial placenta for ex-utero fetal therapies are new frontiers in this exciting field.

Living with osteogenesis imperfecta: A qualitative study exploring experiences and psychosocial impact from the perspective of patients, parents and professionals.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic condition characterised by increased bone fragility. Recurrent fractures, pain and fatigue have a considerable impact on many aspects of the life of a person affected with OI and their families. OBJECTIVE: To improve our understanding of the impact of OI on the daily lives of individuals and families and consider how the condition is managed so that support needs can be better addressed. METHODS: Semi-structured qualitative interviews (n = 56) were conducted with adults affected with OI, with (n = 9) and without children (n = 8), parents of children affected with OI (n = 8), health professionals (n = 29) and patient advocates (n = 2). Interviews were digitally recorded, transcribed verbatim and analysed using thematic analysis. RESULTS: Three overarching themes are described: OI is not just a physical condition, parenting and family functioning and managing the condition. Fractures, chronic pain and tiredness impact on daily life and emotional well-being. For parents with OI, pain, tiredness and mobility issues can limit interactions and activities with their children. Specialist paediatric health services for OI were highly valued. The need for more emotional support and improved coordination of adult health services was highlighted. CONCLUSIONS: Our findings allow a better understanding of the day-to-day experiences of individuals and families affected with OI. Supporting emotional well-being needs greater attention from policy makers and researchers. Improvements to the coordination of health services for adults with OI are needed and an in-depth exploration of young people's support needs is warranted with research focused on support through the teenage years.

Haemoglobin and red blood cell reference intervals during infancy.

OBJECTIVES: There is a need for updated haematological reference data in infancy. This study aimed to define intervals for haemoglobin and red blood cell biomarkers based on data from a large cohort of longitudinally followed Swedish infants. DESIGN: Longitudinal cohort study. SETTING: Two Swedish study centres. PARTICIPANTS: Three community-based populations including 442 presumably healthy infants born at term and with umbilical cord clamping delayed to 30 s or more after birth. METHODS: Blood samples were collected from umbilical cord blood (a), at 48-118 hours (b), at 4 months (c) and at 12 months (d). Reference intervals as the 2.5th and 97.5th percentiles were calculated in coherence with Clinical and Laboratory Standards Institute guidelines. RESULTS: Reference intervals for haemoglobin (g/L) were: (a) 116-189, (b) 147-218, (c) 99-130, (d) 104-134, and for mean cell volume (fL): (a) 97-118, (b) 91-107, (c) 71-85, (d) 70-83. Reference intervals for erythrocyte counts, reticulocyte counts, reticulocyte haemoglobin, mean cell haemoglobin and mean cell haemoglobin concentration were also estimated. According to the WHO definition of anaemia, a haemoglobin value less than 110 g/L, 16% of this presumably healthy cohort could be classified as anaemic at 12 months. CONCLUSION: We found mainly narrower reference intervals compared with previously published studies. The reference intervals for each parameter varied according to the infants' age, demonstrating the necessity of age definitions when presenting infant reference intervals. The discrepancy with the WHO classification for anaemia at 12 months, despite favourable conditions in infancy, needs future investigation.

Mesenchymal Stem Cell Therapy for Osteogenesis Imperfecta.

The aim of this study was to provide a brief overview on the background and rationale on treating fetuses and children suffering from osteogenesis imperfecta (OI) with mesenchymal stem cells (MSCs). MSCs ability to migrate, engraft, and differentiate into bone cells and to act via paracrine effects on the recipient's tissues makes these cells promising candidates as a clinical therapy for OI. Animal work and limited clinical studies in humans support the use of MSC in treating OI. Off-the-shelf MSC have a good safety profile and exhibit multilineage differentiation potential and a low immunogenic profile and thereby may enable this potential therapy to become widely available. MSC transplantation before and after birth to treat OI is an experimental therapy that is currently tested in the international multicentre phase I/II clinical trial BOOSTB4 that aims to assess the safety and efficacy of fetal MSC transplantation for the treatment of severe types of OI.

In Vitro Study of Human Immune Responses to Hyaluronic Acid Hydrogels, Recombinant Spidroins and Human Neural Progenitor Cells of Relevance to Spinal Cord Injury Repair.

Scaffolds of recombinant spider silk protein (spidroin) and hyaluronic acid (HA) hydrogel hold promise in combination with cell therapy for spinal cord injury. However, little is known concerning the human immune response to these biomaterials and grafted human neural stem/progenitor cells (hNPCs). Here, we analyzed short- and long-term in vitro activation of immune cells in human peripheral blood mononuclear cells (hPBMCs) cultured with/without recombinant spidroins, HA hydrogels, and/or allogeneic hNPCs to assess potential host-donor interactions. Viability, proliferation and phenotype of hPBMCs were analyzed using NucleoCounter and flow cytometry. hPBMC viability was confirmed after exposure to the different biomaterials. Short-term (15 h) co-cultures of hPBMCs with spidroins, but not with HA hydrogel, resulted in a significant increase in the proportion of activated CD69+ CD4+ T cells, CD8+ T cells, B cells and NK cells, which likely was caused by residual endotoxins from the Escherichia coli expression system. The observed spidroin-induced hPBMC activation was not altered by hNPCs. It is resource-effective to evaluate human compatibility of novel biomaterials early in development of the production process to, when necessary, make alterations to minimize rejection risk. Here, we present a method to evaluate biomaterials and hPBMC compatibility in conjunction with allogeneic human cells.

Trophic effects of multiple administration of mesenchymal stem cells in children with osteogenesis imperfecta.

The safety of mesenchymal stem cell therapy for osteogenesis imperfecta has been demonstrated previously. However, it is unknown how the trophic effects are mediated by stem cells. In the present commentary, we bring to the attention of readers the recent report by Infante et al in the journal of clinical and translational medicine. The TERCELOI clinical trial presented the possible paracrine effect of transplanted MSCs in vitro and in vivo using proteomics and transcriptomic analysis. This novel finding adds new knowledge in the field of regenerative medicine. However, the scarcity of solid evidence in growth warrants a more thorough discussion.

The Effect of Mesenchymal Stromal Cells Derived From Endometriotic Lesions on Natural Killer Cell Function.

Endometriosis is an inflammatory disease that presents with ectopic endometriotic lesions. Reduced immunosurveillance of these lesions has been proposed to be playing a role in the pathology of endometriosis. Mesenchymal stromal cells (MSC) are found in ectopic lesions and may decrease immunosurveillance. In the present study, we examined if MSC contribute to reduced immunosurveillance through their immunosuppressive effects on natural killer (NK) cells. Stromal cells from endometriotic ovarian cysts (ESCcyst) and eutopic endometrium (ESCendo) of women with endometriosis and their conditioned medium were used in co-cultures with allogeneic peripheral blood NK cells. Following culture, NK cells were examined phenotypically for their expression of activating, inhibitory, maturation, and adhesion receptors and co-receptors, as well as the degranulation (CD107a) marker and the immunostimulatory (interferon-γ) and immunosuppressive (transforming growth factor beta 1 and interleukin-10) cytokines. Moreover, NK cell cytotoxicity was examined using chromium 51 release killing assays. There were no differences between ESCcyst and ESCendo regarding their effects on NK cell cytotoxicity in both conditioned medium and direct co-culture experiments. Additionally, there were no differences between ESCcyst and ESCendo regarding their impact on NK cells' phenotype and degranulation in both conditioned medium and direct co-culture experiments. Although there were no differences found for DNAX accessory molecule-1 (DNAM-1) and NKp44, we found that the expression of the NK cell ligand CD155 that binds DNAM-1 and proliferating cell nuclear antigen (PCNA) that binds NKp44 was significantly less on ESCcyst than on ESCendo. These findings were not supported by the results that the expression of the known and unknown ligands on ESCcyst for DNAM-1 and NKp44 using chimeric proteins was not significantly different compared to ESCendo. In conclusion, the results suggest that ectopic MSC may not contribute to reduced immunosurveillance in endometriosis through their inhibitory effects on NK cells. This suggests that NK cell inhibition in the pelvic cavity of women with endometriosis develops due to other factors.

Soluble Transferrin Receptor during infancy and reference intervals for the Roche Cobas platform.

INTRODUCTION: Infant iron status assessments may be difficult to interpret due to infections. The soluble transferrin receptor (sTfR) has been suggested as a biomarker mainly unaffected by the acute phase response. Reference intervals reflecting dynamics of infant growth first year in life are not well established. METHODS: The sTfR and CRP concentrations were measured in samples from 451 term infants with the Roche Cobas platform in umbilical cord, at 48-96 hours, 4 and 12 months. Reference values were constructed as the 2.5th and 97.5th percentiles. The relationship between CRP concentrations >1 mg/L and sTfR was tested by Kendall correlation. RESULTS: Reference intervals for girls and boys were 2.4-9.5 mg/L at birth, 2.9-8.4 mg/L at 48-96 hours, 2.6-5.7 mg/L at 4 months and 3.0-6.3 mg/L at 12 months. No differences between sexes were observed except for at 4 months. sTfR did not covariate with CRP concentrations >1 mg/L except in 48-96 hours samples. CONCLUSION: This study reports reference intervals for sTfR from birth to 12 months of age in a large group of infants in a low-risk area for iron deficiency. sTfR might add value to infant iron status diagnostics since no covariation with CRP was found at birth, at 4 months or at 12 months.

Stem Cell Therapy as a Treatment for Osteogenesis Imperfecta.

PURPOSE OF REVIEW: Osteogenesis imperfecta (OI) is a chronic disease with few treatment options available. The purpose of this review is to provide an overview on treating OI with mesenchymal stem cells (MSC). RECENT FINDINGS: Off-the-shelf MSC have a good safety profile and exhibit multilineage differentiation potential and a low immunogenic profile and are easy to manufacture. Their ability to migrate, engraft, and differentiate into bone cells, and also to act via paracrine effects on the recipient's tissues, makes MSC candidates as a clinical therapy for OI. Due to their high osteogenic potency, fetal MSC offer an even higher therapeutic potential in OI compared with MSC derived from adult sources. Preclinical and initial clinical data support the use of MSC in treating OI. The characteristics of MSC make them of great interest in treating OI. MSC may be safely transplanted via intravenous administration and show potential positive clinical effects.

Prenatal stem cell therapy for inherited diseases: Past, present, and future treatment strategies.

Imagine the profits in quality of life that can be made by treating inherited diseases early in life, maybe even before birth! Immense cost savings can also be made by treating diseases promptly. Hence, prenatal stem cell therapy holds great promise for developing new and early-stage treatment strategies for several diseases. Successful prenatal stem cell therapy would represent a major step forward in the management of patients with hematological, metabolic, or immunological disorders. However, treatment before birth has several limitations, including ethical issues. In this review, we summarize the past, the present, and the future of prenatal stem cell therapy, which includes an overview of different stem cell types, preclinical studies, and clinical attempts treating various diseases. We also discuss the current challenges and future strategies for prenatal stem cell therapy and also new approaches, which may lead to advancement in the management of patients with severe incurable diseases.

Urothelial cell senescence is not linked with telomere shortening.

The success of regenerative medicine relies in part on the quality of the cells implanted. Cell cultures from cells isolated from bladder washes have been successfully established, but molecular changes and cell characteristics have not been explored in detail. In this work, we analysed the role of telomere shortening in relation to the regenerative potential and senescence of cells isolated from bladder washes and expanded in culture. We also analysed whether bladder washes would be a potential source for attaining stem cells or promoting stem cell proliferation by using two different substrates to support their growth: a feeder layer of growth-arrested murine fibroblasts J2 3T3 cells and a xeno-free human recombinant laminin-coated surface. We found no association between telomere shortening and senescence in urothelial cells in vitro. Urothelial cells had a stable telomere length and expressed mesenchymal stem cells markers but failed to differentiate into bone or adipocytes. Feeder layer showed an advantage to laminin-coated surfaces in respect to proliferative capacity with the expense of risking that feeder layer cells could persist in later passages. This emphasizes the importance of using carefully controlled culture conditions and molecular quality controls before autotransplantation in future clinical settings. In conclusion, urothelial cells isolated by bladder washes show regenerative potential that need further understanding. Senescence in vitro might be due to cellular stress, and if so, further improvements in culture conditions may lead to longer cell life and higher proliferative capacity.

Correction to: Defined serum- and xeno-free cryopreservation of mesenchymal stem cells.

In the original article, Fig. 1A was by mistakenly duplicated. The corrected image is provided in this correction article.

Stakeholder views and attitudes towards prenatal and postnatal transplantation of fetal mesenchymal stem cells to treat Osteogenesis Imperfecta.

The Boost Brittle Bones Before Birth (BOOSTB4) clinical trial is investigating the safety and efficacy of transplanting fetal derived mesenchymal stromal cells (MSCs) prenatally and/or in early postnatal life to treat severe Osteogenesis Imperfecta (OI). This study aimed to explore stakeholder views to understand perceived benefits or concerns, identify ethical issues and establish protocols for support and counselling. Semi-structured qualitative interviews were conducted with three groups; 1. Adults affected with OI, with and without children, and parents of children affected with OI; 2. Health professionals who work with patients with OI; 3. Patient advocates from relevant patient support groups. Interviews were digitally recorded, transcribed verbatim and analysed using thematic analysis. Interviews with 56 participants revealed generally positive views towards using fetal MSC transplantation to treat OI. Early treatment was considered advantageous for preventing fractures and reducing severity and could bring psychological benefits for parents. Common concerns were procedure safety, short/long-term side effects and whether transplantation would be effective. Difficulties inherent in decision-making were frequently discussed, as treatment efficacy is unknown and, by necessity, parents will make decisions at a time when they are vulnerable. Support needs may differ where there is a family history of OI compared to an unexpected diagnosis of OI. Explaining fetal MSC transplantation in a way that all parents can understand, clear expectation setting, psychological support and time for reflection during the decision-making process will be crucial to allow parents to make informed decisions about participation in the BOOSTB4 clinical trial.

Fetal stem cell transplantation and gene therapy.

The present chapter summarizes our current knowledge on fetal stem cell and gene therapy. It focuses on these therapeutic alternatives in regard to past experiences and ongoing and planned studies in humans. Several methodological challenges are discussed that may have wide implications on how these methods could be introduced in clinical practices. Although still promising, the methods are afflicted with very special requirements not least in regard to safety and ethical questions. Furthermore, careful monitoring and extended follow-up of the child and his/hers mother who receive prenatal stem cell or gene treatments are of outmost importance. Taken these prerequisites into consideration, it is natural that this type of experimental fetal therapies requires collaboration between different disciplinaries and institutions within medicine.

Exploring the impact of Osteogenesis Imperfecta on families: A mixed-methods systematic review.

BACKGROUND: Osteogenesis Imperfecta (OI) is a rare genetic condition whose key characteristic is increased bone fragility. OI has the potential to impact upon all family members, making it important to consider the challenges families face, how they cope and their support needs as the affected individual moves from childhood through to adult life. OBJECTIVE: To conduct a mixed-methods systematic review investigating the experiences of families when a family member is affected with OI. METHODS: A systematic search of seven electronic databases, relevant patient organisation websites and reference lists was conducted. Data extraction was performed for all studies that met the eligibility and quality criteria. Results were synthesised following the principles of thematic analysis. RESULTS: One mixed-method, six qualitative and six quantitative studies were included in the review. Three overarching themes were identified through thematic analysis: Impact of OI on the psychosocial wellbeing of families, impact on family life and evolving roles and relationships. Fear of fractures and the uncertainty of when the next fracture will occur are key issues that permeate all areas of family life and impact upon all family members. CONCLUSION: The experiences, coping strategies and support needs of families affected by OI were highly variable and changed over time. Future research should address the need for adaptive health and education interventions that support all family members.